High throughput screening (HTS) of protein targets for drug discovery is a very costly process. On average it cost about $100 million dollars to develop one drug and only three percent of the drugs make it through phase I clinical trials. About 5-15% of all proteins targets that are chosen for HTS never make it into the pipeline because the proteins are either insoluble, inactive, or unstable (sensitive to proteolysis). Although yeast and baculovirus systems for gene expression are often used as reliable alternatives, bacterial expression systems are still the main method of choice for over expressing recombinant proteins for drug development studies.
Protein insolubility is one of the major problems associated with over-expressing proteins in bacteria. Protein solubility is judged empirically by assaying the levels of recombinant protein in the supernatant and pellet of lysed protein extracts. In general, small proteins (>30 kD) that are simple monomeric proteins can be found in the soluble fractions of bacterial extracts. In contrasts, proteins (<30 kD) or proteins that have complex secondary or tertiary structures are typically insoluble and are predominantly found in inclusion bodies. Although insoluble proteins can be denatured in urea and refolded in situ, full activity frequently is not restored.
Yet another problem associated with the recombinant expression of proteins is proteolysis in vivo. Although a number of proteases have been removed by gene knock out technologies to reduce proteolysis in bacterial expression systems (such as Ion in BL21 DE3), many proteins are very sensitive to endogenous bacterial proteases. Sensitivity to proteases can dramatically reduce the yield of full-length recombinant proteins. In extreme cases the expression of recombinant proteins is nearly undetectable due to proteolysis.